Over the last few years, in-vivo scanning using Positron Emission Tomography (PET) has increased. PET is both a medical and research tool. It is used heavily in clinical oncology for medical imaging of tumors and the search for metastasis, and for clinical diagnosis of certain diffuse brain diseases such as those causing various types of dementias. Radiotracers consisting of a radionuclide stably bound to a biomolecule are used for in vivo imaging of disorders.
In designing an effective radiopharmaceutical tracer for use as a diagnostic agent, it is imperative that the drug has appropriate in vivo targeting and pharmacokinetic properties. Fritzberg et al. (J. Nucl. Med., 1992, 33:394) state further that radionuclide chemistry and associated linkages underscore the need to optimize the attachment and labelling of chemical modifications of the biomolecule carrier, diluent, excipient or adjuvant. Hence the type of radionuclide, the type of biomolecule and the method used for linking them to one another may have a crucial effect onto the radiotracer properties.
Peptides are biomolecules that play a crucial role in many physiological processes including actions as neurotransmitters, hormones, and antibiotics. Research has shown their importance in such fields as neuroscience, immunology, pharmacology and cell biology. Some peptides can act as chemical messenger. They bind to receptor on the target cell surface and the biological effect of the ligand is transmitted to the target tissue. Hence the specific receptor binding property of the ligand can be exploited by labelling the ligand with a radionuclide. Theoretically, the high affinity of the ligand for the receptor facilitates retention of the radio labeled ligand in receptor expressing tissues. However, it is still under investigation which peptides can efficiently be labeled and under which conditions the labelling shall occur. It is well known that receptor specificity of ligand peptide may be altered during chemical reaction. Therefore an optimal peptidic construct has to be determined.
Tumors overexpress various receptor types to which peptide bound specifically. Boerman et al. (Seminar in Nuclear Medicine, 30(3) July, 2000; pp 195-208) provide a non exhaustive list of peptides binding to receptor involved in tumor, i.e., somatostatin, vasoactive intestinal peptide (VIP), bombesin binding to gastrin-releasing peptide (GRP) receptor, gastrin, cholecystokinin (CCK) and calcitonin.
The radionuclides used in PET scanning are typically isotopes with short half lives such as 11C (˜20 min), 13N (˜10 min), 15O (˜2 min), 68Ga (˜68 min) or 18F (˜110 min). Due to their short half lives, the radionuclides must be produced in a cyclotron which is not too far away in delivery-time from the PET scanner. These radionuclides are incorporated into biologically active compounds or biomolecules that have the function to vehicle the radionuclide into the body though the targeted site, for example a tumor.
The linkage of the radionuclide to the biomolecule is done by various methods resulting in the presence or not of a linker between the radionuclide and the biomolecule. Hence, various linkers are known. C. J. Smith et al. (“Radiochemical investigations of 177Lu-DOTA-8-Aoc-BBN[7-14]NH2: an in vitro/in vivo assessment of the targeting ability of this new radiopharmaceutical for PC-3 human prostate cancer cells.” Nucl. Med. Bio., 30(2):101-9; 2003) disclose radiolabeled bombesin wherein the linker is DOTA-X where X is a carbon tether. However, the radiolabel 177Lu (half life 6.5 days) does not match the biological half-life of the native bombesin what makes the 177Lu-DOTA-X-bombesin a non-appropriate radiotracer for imaging tumor.
E. Garcia Garayoa et al. (“Chemical and biological characterization of new Re(CO)3/[99mTc](CO)3 bombesin Analogues.” Nucl. Med. Biol., 17-28; 2007) disclose a spacer between the radionuclide [99mTc] and the bombesin wherein the spacer is -β-Ala-β-Ala- and 3,6-dioxa-8-aminooctanoic acid. E. Garcia Garayoa et al. conclude that the different spacer does not have a significant effect on stability or on receptor affinity.
Listed above linkers have been specifically designed for a specific type of radionuclide and determine the type and chemical conditions of the radiobinding method.
More recently, peptides have been conjugated to a macrocyclic chelator for labelling with 64Cu, 86Y, and 68Ga for PET application. However, such radionuclides interact with the in vivo catabolism resulting in unwanted physiologic effects and chelate attachment.
The nucleophilic aromatic 18F-fluorination reaction is of great importance for 18F-labeled radiopharmaceuticals which are used as in vivo imaging agents for targeting and visualizing diseases, e.g., solid tumors or diseases of brain. A very important technical goal in using 18F-labeled radiopharmaceuticals is the quick preparation and administration of the radioactive compound due to the fact that the 18F isotopes have a short half-life of about only 111 minutes.
18F-labeled compounds are gaining importance due to the availability thereof as well as due to the development of methods for labeling biomolecules. It has been shown that some compounds labeled with 18F produce images of high quality. Additionally, the longer lifetime of 18F would permit longer imaging times and allow preparation of radiotracer batches for multiple patients and delivery of the tracer to other facilities, making the technique more widely available to clinical investigators. Additionally, it has been observed that the development of PET cameras and availability of the instrumentation in many PET centers is increasing. Hence, it is increasingly important to develop new tracers labeled with 18F.
The nucleophilic aromatic 18F-fluorination reaction is of great importance for 18F-labeled radiopharmaceuticals which are used as in vivo imaging agents targeting and visualizing diseases, e.g., solid tumors.
Various methods of radiofluorination have been published using different precursors or starting material for obtaining 18F-labeled peptides. Due to the smaller size of peptides, both higher target-to-background ratios and rapid blood clearance can often be achieved with radiolabeled peptides. Hence, short-lived positron emission tomography (PET) isotopes are potential candidates for labelling peptides. Among a number of positron-emitting nuclides, fluorine-18 appears to be the best candidate for labelling bioactive peptides by virtue of its favourable physical and nuclear characteristics. The major disadvantage of labelling peptides with 18F is the laborious and time-consuming preparation of the 18F labelling agents. Due to the complex nature of peptides and several functional groups associated with the primary structure, 18F-labeled peptides are not prepared by direct fluorination. Hence, difficulties associated with the preparation of 18F-labeled peptide were alleviated with the employment of prosthetic groups as shown below. Several such prosthetic groups have been proposed in the literature, including N-succinimidyl-4-[18F]fluorobenzoate, m-maleimido-N-(p-[18F]fluorobenzyl)-benzamide, N-(p-[18F]fluorophenyl)maleimide, and 4-[18F]fluorophenacylbromide. Almost all of the methodologies currently used today for the labeling of peptides and proteins with 18F utilize active esters of the fluorine labeled synthon.

Okarvi et al. (“Recent progress in fluorine-18 labeled peptide radiopharmaceuticals.” Eur. J. Nucl. Med., 2001 July; 28(7):929-38)) present a review of the recent developments in 18F-labeled biologically active peptides used in PET.
Xianzhong Zhang et al. (“18F-labeled bombesin analogs for targeting GRP receptor-expressing prostate cancer.” J. Nucl. Med., 47(3):492-501 (2006)) relate to the 2-step method detailed above. [Lys3]Bombesin ([Lys3]BBN) and aminocaproic acid-bombesin(7-14) (Aca-BBN(7-14)) were labeled with 18F by coupling the Lys3 amino group and Aca amino group, respectively, with N-succinimidyl-4-18F-fluorobenzoate (18F-SFB) under slightly basic condition (pH 8.5). Unfortunately, the obtained 18F-FB-[Lys3]BBN is metabolically relatively unstable having for result to reduce the extent of use of the 18F-FB-[Lys3]BBN for reliable imaging of tumor.
Thorsten Poethko et al. (“Two-step methodology for high-yield routine radiohalogenation of peptides: 18F-labeled RGD and octreotide analogs.” J. Nucl. Med., 2004 May; 45(5):892-902) relate to a 2-step method for labelling RGD and octreotide analogs. The method discloses the steps of radiosynthesis of the 18F-labeled aldehyde or ketone and the chemoselective ligation of the 18F-labeled aldehyde or ketone to the aminooxy functionalized peptide.
Thorsten Poethko et al. (“First 18F-labeled tracer suitable for routine clinical imaging of somatostatin receptor-expressing tumors using positron emission tomography.” Clin. Cancer Res., 2004 Jun. 1; 10(11):3593-606) apply the 2-step method for the synthesis of 18F-labeled carbohydrated Tyr(3)-octreotate (TOCA) analogs with optimized pharmacokinetics suitable for clinical routine somatostatin-receptor (sst) imaging.
WO 2003/080544 A1 and WO 2004/080492 A1 relate to radiofluorination methods of bioactive peptides for diagnostics imaging using the 2-step method shown above.
The most crucial aspect in the successful treatment of any cancer is early detection. Likewise, it is crucial to properly diagnose the tumor and metastasis.
Routine application of 18F-labeled peptides for quantitative in vivo receptor imaging of receptor-expressing tissues and quantification of receptor status using PET is limited by the lack of appropriate radiofluorination methods for routine large-scale synthesis of 18F-labeled peptides. There is a clear need for radiofluorination method that can be conducted rapidly without loss of receptor affinity by the peptide and leading to a positive imaging (with reduced background), wherein the radiotracer is stable and shows enhanced clearance properties
The conversions of mono- (mainly para-) substituted trimethylammonium benzene derivatives (1) to substituted [18F]-fluoro-benzene derivatives (2) which may serve as radiopharmaceutical itself or as prosthetic group for the F-18 labeling of small and large molecules have been reported in the literature (We et al. 1982, Fluorine Chem., 27, (1985), 117-191; Haka et al. 1989) (see Scheme 1).

There are only a few publications about nucleophilic aromatic 18F-fluorination reactions of trimethyl ammonium substituted aromatic derivatives which contain two or more substituents beside the trimethylammonium moiety:
Oya et al. treated [2-Chloro-5-(2-dimethylcarbamoyl-phenylsulfanyl)-4-nitro-phenyl]-trimethyl-ammonium triflate with [18F]potassium fluoride and obtained the desired 18F-labeled compound (J. Med. Chem., 2002, 45(21):4716-4723).
Li et al. report on the 18F-fluorination reaction of 4-(N,N,N-trimethylammonium)-3-cyano-3′-iodobenzophenone triflate (Bioconjugate Chemistry, 2003, 14(2):287-294).
Enas et al. convert (2,2-Dimethyl-1,3-dioxo-indan-5-yl)-trimethyl-ammonium triflate into the desired 18F-labeled compound (J. Fluorine Chem., 1993, 63(3):233-41).
Seimbille et al. and other groups have labeled (2-Chloro-4-nitro-phenyl)-trimethyl-ammonium triflate successfully with 18F (J. Labeled Compd. Radiopharm., 2005, 48, 11:829-843).
(2-Benzyloxy-4-formyl-phenyl)-trimethyl-ammonium triflate has successfully been labeled with 18F at high temperature (130° C.) by Langer et. al. (Bioorg. Med. Chem., EN; 9; 3; 2001:677-694).
Lang et al. have radiolabeled trimethyl-(2-methyl-4-pentamethylphenyl methoxycarbonyl-phenyl)-ammonium triflate by using [18F]potassium fluoride (J. Med. Chem., 42, 9, 1999:1576-1586).
Trimethyl-(4-nitro-naphthalen-1-yl)-ammonium triflate has been labeled with 18F by Amokhtari et al. (J. Labeled Compd. Radiopharm.; S42, 1 (1999):S622-S623).
Lemaire et al. have converted (2-formyl-5-methoxy-phenyl)-trimethyl-ammonium triflate into the desired 18F-labeled product (J. Labeled Compd. Radiopharm., 44, 2001:S857-S859).
VanBrocklin et al. describe the 18F labeling of (2-bromo-4-nitro-phenyl)-trimethyl-ammonium triflate (J. Labeled Compd. Radiopharm., 44; 2001:S880-S882) and Cetir Centre Medic report on the successful 18F-labeling of (5-Chloro-8-hydroxy-quinolin-7-yl)-trimethyl-ammonium triflate (EP 1 563 852 A1).
D. A. Sutton et al. (“Evaluation of 1-fluoro-2-nitro-4-trimethylammoniobenzene iodide, a protein-solubilizing agent”, Biochem. J., 1972, 130:589-595) disclose model derivatives which consist of a benzene substituted with trimethylammonium, an electron withdrawing nitro group and a glycine, phenylalanine or acetyltyrosine.
C. Lemaire et al. (“Highly enantioselective synthesis of no-carrier-added 6-[18F]fluoro-L-dopa by chiral phase-transfer alkylation”, Eur. J. Org. Chem., 2004:2899-2904) disclose 2-[18F]fluoro-4,5-dimethoxybenzaldehyde to be used to prepare 6-[18F]fluoro-L-dopa.
L. Lang et al. (“Development of fluorine-18-labeled 5-HT1A antagonists”, J. Med. Chem., 1999, 42(9):1576-1586) disclose conversion of pentamethyl 4-(trimethylammonium trifluormethanesulfonate)benzoate and pentamethyl 3-methyl-4-(trimethylammonium trifluormethansulfonate)benzoate to the respective 18F substituted benzoyl chloride which is then coupled with WAY 100635 (N-{2-[4-(2-methoxyphenyl)-piperazino]ethyl}-N-(2-pyridyl)cyclohexanecarboxamide).
S. Oya et al. (“New PET imaging agent for the serotonin transporter: [18F]ACF (2-[(-amino-4-chloro-5-fluorophenyl)thio]-N,N-dimethyl-benzenmethanamine)”, J. Med. Chem., 2002, 45:4716-4723) disclose conversion of [2-chloro-5-(2-dimethylaminocarbonyl-phenylthio)-4-nitro-phenyl]trimethylammonium trifluoromethanesulfonate to the respective 18F substituted compound.
M. J. Al-Darwich et al. (“Enantioselective synthesis of non-carrier-added (n.c.a.) (S)-4-chloro-2-[18F]fluorophenylalanine and (S)-(α-methyl)-4-chloro-2-[18F]fluorophenylalanine”, J. Fluorine Chem., 1996, 80:117-124) disclose 4-chloro-2-trimethylammoniumbenzaldehyde triflate to be reacted to 4-chloro-2-[18F]fluorobenzaldehyde which are then further reacted to yield the title compounds.
Y. Seimbille et al. (“Fluorine-18 labeling of 6,7-disubstituted anilinoquinazoline derivatives for positron emission tomography (PET) imaging of tyrosine kinase receptors: synthesis of 18F-Iressa and related molecular probes”, J. Labeled Compd. Radiopharm., 2005, 48:829-843, i.a., report on the reaction of 3-chloro-4-trimethylammonium nitrobenzene trifluoro-methanesulfonate to give 3-chloro-4-[18F]fluoroaniline via 3-chloro-4-[18F]fluoro-nitro-benzene.
WO 2002/44144 A1 relates to nucleophilic reaction for preparing radiolabeled imaging agents using [18F]fluoride to react with trimethylammoniumbenzene compounds.
WO 2006/083424 A2 relates to [18F]-radiolabeled compounds and the manufacture thereof.
Most of these mentioned 18F-labeled aromatic derivatives which contain two or more additional substituents cannot be coupled to chemical functionalities like amines, thiols, carboxylic acids, phenols or other chemicals groups of complex molecules like peptides without further transformations.
18F labeling of more complex radiopharmaceuticals like peptides takes place in all known publications in a two- or multi-step strategy (see Scheme 2, review article: Eur. J. Nucl. Med., 2001, 28:929-938).
For these kinds of 18F-labeling also mono-substituted trimethylammonium benzene derivatives are used and react in a first step with [18F]potassium fluoride to obtain substituted [18F]-fluoro-benzene derivatives. These compounds are then coupled in a second step to larger and more complex molecules like peptides, small molecules or nucleotides (see scheme 2).

Especially 4-[18F]fluorobenzaldehyde has been used in many examples for F-18 labelling of complex molecules (e.g., J. Nucl. Med., 2004, 45(5):892-902). But also N-succinimidyl-8-[4′-[18F]fluorobenzylamino]suberate (Bioconjugate Chem., 1991, 2:44-49), 4-[18F]fluorophenacyl bromide and 3-[18F]fluoro-5-nitrobenzimidate (J. Nucl. Med., 1987, 28:462-470), m-maleimido-N-(p-[18F]fluorobenzyl)-benzamide (J. Labeled Compd. Radiopharm., 1989, 26:287-289,), N-{4-[4-[18F]fluorobenzylidene(aminooxy)-butyl}-maleimide (Bioconjugate Chem., 2003, 14:1253-1259), [18F]N-(4-fluorobenzyl)-2-bromoacetamide (Bioconjugate Chem., 2000, 11:627-636) and [18F]-3,5-difluorophenyl azide (and 5 derivatives) (J. Org. Chem., 1995, 60:6680-6681) are known examples. F-18 labeling of peptides via para-[18F]-fluorobenzoates is also a very common method either by coupling of the corresponding acid with additional activating agents (such as 1,3-dicyclohexylcarbodiimide/1-hydroxy-7-azabenzotriazole (DCC/HOAt) or N-[(dimethyl-amino)-1H-1,2,3-triazolyl[4,5]pyridine-1-yl-methylene]-N-methyl-methan-aminium hexafluorophosphate N-oxide (HATU/DIPEA, Eur. J. Nucl. Med. Mol. Imaging., 2002, 29:754-759) or by isolated N-succinimidyl 4-[18F]fluorobenzoate (Nucl. Med. Biol., 1996, 23:365).
None of these compounds and none of other published compounds allow a direct (one-step) labeling of peptides with 18F-fluoride.
Therefore is an object of the present invention the development of a practical and mild technique for 18F labeling of molecules like, e.g., peptides, oligonucleotides or small molecule targeting agents and to provide radiofluorination methods for obtaining radiotracer based on receptor specific peptides for the detection of tumors.